We are investigating the molecular mechanisms which control the individual and total concentrations of hemoglobins in human erythrocytes. In addition, we are studying the effects of functional alpha globin gene number, fetal hemoglobin (HbF) levels and the extent of red cell heterogeneity on the various manifestations of sickle cell disease and its genetic variants. The levels of each of the normal hemoglobins (A, A2, F) are determined by controls at the level of transcription and/or translation of the globin genes, as well as by factors that regulate protein degradation. The study of the control of hemoglobin levels has direct relevance to various hemoglobinopathies, especially thalassemia and sickle cell disease. For our experimental system, we are using the K562 human leukemic cell line, as well as peripheral blood from individuals with sickle cell disease. We are studying the effects of short-term and long- term exposure of these cells to 5-azacytidine ane hemin on their phenotype and the factors that control globin gene transcription. Adult beta-mRNA expression remains undetectable, yet we have found a constitutive level of another adult type hemoglobin, delta-mRNA, whose expression is inducible both with hemin and 5-azacytidine. Because of the close sequence homologies between the delta- and beta-globin genes, experiments are underway to examine whether changes in the delta-promoter sequence may alter important protein binding sites and thereby result in the low levels of delta-globin gene expression. Concurrently, we are also attempting to develop a sickle cell mouse model by the introduction of a cloned human sickle globin gene into the mouse germ line by the microinjection of DNA into the pronuclei of fertilized eggs. The establishment of such a model would allow for basic and fundamental questions to be asked about the molecular, cellular and physiologic aspects of the disease, as well as provide an in vivo system to monitor the effects of potential therapy.